CA1335577C

CA1335577C - Superoxide dismutase analogs having novel binding properties

Info

Publication number: CA1335577C
Application number: CA000602018A
Authority: CA
Inventors: Guy T. Mullenbach; Robert A. Hallewell; Pablo Valenzuela
Original assignee: Chiron Corp
Current assignee: Novartis Vaccines and Diagnostics Inc
Priority date: 1988-06-14
Filing date: 1989-06-07
Publication date: 1995-05-16
Anticipated expiration: 2012-05-16
Also published as: JPH03505398A; DE68923613D1; ATE125566T1; JP2942578B2; WO1989012677A1; EP0416037A1; EP0416037B1; DE68923613T2; EP0416037A4

Abstract

Superoxide dismutase (SOD) analogs whose amino acid sequence includes a heterologous peptide domain such as the RGDX tetrapeptide found in fibronectin or the LGGAKQAGDV decapeptide found in fibrinogen that enables the analog to bind to a moiety that is normally present at sites of desired SOD therapy. The domain is incorporated into the SOD sequence in a manner in which it is available to interact with such moieties but does not destroy the enzymatic activity of the molecule. The inclusion of the domain enables the analog to more readily access the target tissue upon which it is to act and/or increase the corporeal half-life of the molecule.

Description

1 3355~7 SUPEROXIDE DISMUTASE ANALOGS
HAVING NOVEL BINDING PROPERTIES

Description Technical Field This invention is in the fields of protein chemistry, genetic engineering, and pharmaceuticals. More particularly, it relates to superoxide dismutase (SOD) analogs that are modified in a manner in which they have binding properties that enable them to be localized at sites where superoxide dismutase therapy is required and/
or extend their corporeal lifetimes.

Background Art SODs are a family of enzymes that catalyze the destruction of superoxide ions. SODs are metalloproteins that are individually characterized by their respective metal ions--which can be iron, manganese, or copper and zinc. The ability of SODs to catalyze the destruction of superoxide ions renders them useful in a variety of therapeutic settings such as in reducing reperfusion injury and in treating inflammation. The amino acid sequence of human Cu/Zn SOD is described in Jabusch et al, Biochemistry (1980) 19:2310-2316. The cloning and sequencing of human Cu/Zn SOD and the production of human Cu/Zn SOD in bacteria and yeast are described in EPA
84111416.8 (published 24 April 1985 under number 0138111).
Human Cu/Zn SOD is normally a homodimer of two chains bound together by hydrophobic interaction. The homodimer has a molecular weight of approximately 32 kd.
The primary structure of human Mn SOD is described by Barra et al, J.B.C. (1984) 259:12595-12601. The cloning and sequencing of human Mn SOD and the recombinant production of human Mn SOD is described in Belgian Patent No. 905,796 issued 16 March 1987.
In achieving its therapeutic potential SOD must (1) achieve access of the target tissue upon which it is to act (2) maintain its molecular integrity while in transit and at the site of action and (3) not be cleared too rapidly from circulation. Prior modifications of SOD have focused on only the latter two of these requirements. For example, commonly EPA 0 283 244 published 21 September 1988 describes SOD
polymers composed of covalently coupled SOD dimers which are not cleared as rapidly from circulation as native SOD. Res.
Commun. in Chem. Path. & Pharmacol. (1980) 29:113-120 and Proc. Nat'l Acad. Sci. USA (1980) 77:1159-1163 describe conjugates of SOD and macromolecules such as polyethylene glycol that also have increased circulatory half-lives relative to wild-type SOD. J. Clin Invest. (1984) 73:87-95 describes SOD encapsulated in liposomes to protect the SOD
from proteolysis and extend its clearance time.
In contrast, the present invention is primarily directed to modifying SOD in a manner to enable it to more readily access the target tissue upon which it is to act and/or to extend its corporeal lifetime. This is achieved by modifying the amino acid sequence of SOD to include a heterologous peptide domain that binds to a moiety that localizes at the site where the superoxide dismutase therapy is desired or binds to the site iteself. Such domains are sometimes referred to as "adhesive peptide signals" herein.

~.

Prior workers have identified adhesive peptide signals of various proteins that bind to cells, bodies, or molecules that are/real to or commonly localize at sites at which SOD therapy may be desirable. The signals for fibrinogen, fibronectin, and von Willebrand factor are described in Nature (1984) 309:30-33; Proc. Nat'l Acad.
Sci. USA (1984) 81:4935-4939; Cell (1986) 44:517-518 and Cell (1987) 48:867-873. Signals for laminin are described in Science 1987) 238:1132, for extracellular superoxide dismutase (EC-SOD) in PNAS (1987) 84:6340, for platelet-derived growth factor (PDGF) in Nature (1986) 320:695, for tissue plasminogen activator (tPA) in Abstracts 1141, 1144, 1043, and 1587 in "Thrombosis and Haemostasis, Volume 58, Abstracts", Eleventh International Congress, Brussels, Belgium, July 1987, and chemotactic factor in PNAS (1987) 84:9233. Those for Antithrombin III are described in Abstracts 543-545, ibid, and in J. Biol.
Chem. (1987) 262:8061, 11964, 17356. The adhesive peptide signals for anti-platelet GPIIb/IIIa receptor and anti-endothelial cell GPIIb/IIIa receptor antibodies are discussed in Abstracts 15, 715, 901 and 908 of "Thrombosis and Haemostasis, Volume 58, Abstracts", supra. Those for vitronectin appear in Abstracts 15-17 and 564, ibid., for urokinase in Abstract 1140, ibid., for thrombin inhibitor in Abstract 649, ibid. The signal for chemotactic inhibitor is disclosed in Science (1979) 203:461, and for chemotactic agonists in PNAS (1987) 84:7964. The signals for platelet factor-4 appear in Blood (1979) 53:604 and Biochem. J. (1980) 191:769 and those for Gamma IP10 in Nature (1985) 315:672.
Applicants are unaware of any prior efforts to incorporate any of these adhesive domains or signals into other polypeptides to impart binding activity thereto.

Disclosure of the Invention One aspect of this invention is a SOD analog having dismutase activity and whose amino acid sequence includes a heterologous peptide domain that provides the analog with an ability to bind to a moiety that localizes at or is local to a body site at which SOD therapy is desired.
DNA sequences encoding such SOD analogs, expres-sion vectors containing such DNA sequences, cellstransformed with such vectors, and processes for making the analogs by growing such transformed cells are another aspect of the invention.
Still other aspects of the invention are pharmaceutical compositions which contain the analogs and therapeutic methods which employ the analogs.

Brief Description of the Drawings In the drawings:
Figure 1 is the nucleotide sequence of a partially synthetic human Cu/Zn SOD cDNA and the deduced amino acid sequence of human Cu/Zn SOD.
Figure 2 is a schematic illustration of the tertiary structure of human Cu/Zn SOD.
Figure 3 is a flow diagram depicting the scheme used to construct the yeast expression vector pYSOD-AS-T-2.
Figure 4 shows the DNA sequence of the insert used in the construction of pYSOD-AS-T-2.
Figure 5 is a flow diagram depicting the scheme used to construct the yeast expression vector pYSOD-AS-T-8.
Figure 6 shows the DNA sequence of an insert used in the construction of pYSOD-AS-T-8.
Figure 7 is a flow diagram depicting the scheme used to construct the yeast expression vector pYSOD-H-T8.

_5_ t 335577 Figure 8 is a map of the plasmid pNco5AHSODm.
Figure 9 is a flow diagram showing the scheme used to construct pNco5AHSODm.
Figure 10 is a map of the plasmid pGAPXSSHSODm.
Figure 11 is a flow diagram showing the scheme used to construct pPGAPXSSHSODm.

Modes for Carrying Out the Invention A. Definitions The term "SOD" intends a polypeptide having the amino acid sequence of a native intracellular superoxide dismutase and fragments, analogs, or muteins thereof having substantially homologous amino acid sequences thereto which may difer in one or more amino acid substitutions, additions or deletions (other than those directed by the present invention) but still retain the enzymatic activity of the native superoxide dismutase. Examples of such analogs are those described in commonly owned EPA 0 275 202 published 20 July 1988. Polymers of SOD, such as those described in 0 283 244 published 21 September 1988, are intended to be included within the term. The term also encompasses SOD of various mammalian species. Human SOD is preferred for use in humans. Human Cu/Zn SOD and human Mn SOD are preferred species of SOD for modification in accordance with the present invention. The term "substantially homologous", as used above, intends at least about 75% identity, more usùally at least about 85% identity, in amino acid sequence.
A "replicon" is any genetic element (e.g. a plasmid, a chromosome, a virus) that behaves as an autonomous unit of polynucleotide replication within a cell; i.e., capable of replication under its own control.
A "vector" is a replicon in which another polynucleotide segment is attached, so as to bring about the replication and/or expression of the attached segment.
.~

An "expression vector~ refers to a vector capable of autonomous replication or integration and contains control sequences which direct the transcription and translation of the SOD analog DNA in an appropriate host.
A "coding sequence" is a polynucleotide sequence which is transcribed and/or translated into a polypeptide.
A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (i.e., in the 3' direction) coding sequence.
A coding sequence is "under the control" of the promoter sequence in a cell when transcription of the cod-ing sequence results from the binding of RNA polymerase to the promoter sequence; translation of the resulting mRNA
then results in the polypeptide encoded within the coding sequence.
"Operably linked" refers to a juxtaposition wherein the components are configured so as to perform their usual function. Thus, control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
"Control sequences" refers to those sequences which control the transcription and/or translation of the coding sequence(s); these may include, but are not limited to, promoter sequences, transcriptional initiation and termination sequences, and translational initiation and termination sequences. In addition, ~'control sequences"
refers to sequences which control the processing of the polypeptide encoded within the coding sequence; these may include, but are not limited to sequences controlling secretion, protease cleavage, and glycosylation of the polypeptide.
"Transformation~ is the insertion of an exogenous polynucleotide into a host cell. The exogenous polynucleotide may be maintained as a plasmid, or alternatively, may be integrated within the host genome.

B. Characterization of SOD Analogs The amino acid sequence of the SOD analogs of this invention comprise the amino acid sequence of SOD
altered via amino acid substitution, deletion, addition, or combinations thereof to include one or more heterologous peptide domains that provide the analog with the ability to bind to a moiety that localizes at or is local to a body site (e.g., the endothelial wall to which a hemostatic fibrin plug adheres) at which SOD therapy is desired. The binding activity of these analogs lessens the likelihood of their being cleared from circulation.
Accordingly the corporeal half-lives of the analogs will typically be greater than native SOD and the concentration of analog at the desired site maintained at higher levels relative to native SOD.
The moiety to which the analog is capable of binding via the domain(s) depends upon the nature of the domains and may be, for example, cells such as phagocytes (macrophages, neutrophils), fibroblasts and endothelial cells, noncellular bodies such as platelets, or other proteinaceous or nonproteinaceous molecules such as glycosaminoglycans (heparin), fibrin, and thrombin. In some instances these moieties localize at sites of desired SOD therapy (e.g., platelets, neutrophils) or are cells that are local to such a site (e.g., endothelial cells).
The nature and location of the alteration is such that the domain(s) is(are) available to interact with and bind to such moieties but not destroy the superoxide dismutase activity of the analog. Accordingly, the domains will be positioned at a location that is exposed in the tertiary structure of native SOD, such as a hydrophilic loop or the amino or carboxy termini, and their size, charge, hydrophilicity/hydrophobicity, will be such as to not alter the molecule (e.g., by changing its tertiary structure or charge distribution) in a manner that eliminates superoxide dismutase activity. The domain may include innocuous additional amino acid modifications beyond those that are crucial for binding activity. These additional modifications may serve a spacer function or otherwise serve to enhance the accessibility of the domain to the moiety or maintain the enzymatic activity of the analog. Normally the number of amino acid alterations directed by the invention will not exceed about 40, usu-ally not more than about 30, and preferably not more than about 20.
Representative domains are those described in the Background section, supra. In addition, suitable domains that provide binding activity may be identified by making random amino acid alterations at selected sites (e.g, the hydrophilic loops or amino or carboxy termini) in the native SOD sequence and then screening the result-ing analogs for enzymatic activity and binding activity.
Preferred domains are the RGDX tetrapeptide (Nature (1984) 309:30 and U.S. Patent No. 4,578,079), the RGDX
tetrapeptide with up to about a total of 15 to 20 flanking residues (on the carboxy and/or amino terminal side of the tetrapeptide), the decapeptide LGGAKQAGDV which is found at the carboxy terminus of fibrinogen, and that decapeptide with up to a total of about 10 flanking residues (on the carboxy and/or amino terminal side) of the decapeptide found in the native fibrinogen sequence.
"X" in RGDX represents an amino acid that does not eliminate the binding properties of the domain.
It should be appreciated that many of the pos-sible analogs that may be made by incorporating the domains described in the Background section into SOD or by making random alterations in the SOD sequence will either lack enzymatic activity and/or lack the desired binding activity and thus not be within the invention. Those analogs falling within the invention may be identified by routine screening for enzymatic activity and binding activity. Enzymatic activity may be assayed by the pyrogallol assay and binding activity (depending upon the target moiety) according to the various art assays used to identify the domains described in the Background section (e.g., PNAS (1981) 78:2403-2406 and Nature (1984) 309:30-33).
The N-terminus of the analog may be acetylated (as in native human Cu/Zn SOD) or lack acetylation depend-ing upon the organism in which the analog is produced.
Bacterially produced analog will lack such acetylation whereas analog produced in yeast using the procedures described in EPA 0138111 are so acetylated. Analogs made in mammalian cells will also be acetylated. Analogs hav-ing such acetylation are preferred. Similarly, the analog may be glycosylated or unglycosylated depending upon the organism and signaling sequence with which it is produced.

C. Synthesis of SOD Analogs Genes encoding the analogs of human Cu/Zn SOD
may be made via oligonucleotide synthesis and ligation, site directed mutagenesis of the DNA sequences shown in Figure 1 and/or by insertion of synthetic DNA fragments that encode the desired amino acid modifications into a DNA sequence encoding human Cu/Zn SOD. Genes encoding analogs of other SODs, such as Mn SOD, may be made by similar procedures. Site directed mutagenesis techniques are well known in the art. See for instance, Smith and Gilliam in Genetic Engineering Principles and Methods, Plenum press (1981) 3:1-32; Zoller and Smith, Nucleic Acids Res. (1982) 10:6487-6500; and Brake et al, Proc.
Natl. Acad. Sci. USA (1984) 81:4642-4646. The mutant genes may be inserted into suitable prokaryotic or eukaryotic vectors, the resulting expression vectors in-corporated into suitable host organisms or cells, the re-combinant organism or cell grown under conditions thatresult in expression of the mutant gene, and the resulting analog isolated from the host or, if secreted, from the growth medium using the same techniques as are described in said EPA 0138111 to produce recombinant human Cu/Zn SOD.
The metal ions required for enzymatic activity may be provided by growing the recombinant hosts in medium supplemented with the ion(s) or by dialyzing a solution of the analog against a solution containing the ions.
In creating an expression vector, the mutant sequence is located in the vector with the appropriate control DNA sequences, which include a promoter, a ribosomal binding site, and transcriptional and translational stop codons. The positioning and orienta-tion of the coding sequence with respect to the controlsequences is such that the coding sequence is transcribed under the control of the control sequences. In addition to control sequence, it may be desirable to add regulatory sequences which allow for regulation of the expression of the hSOD analog gene relative to the growth of the host cell.

D. Formulation and Use of SOD Analogs The SOD analogs of the invention may be used for the same purposes as SOD. The analogs may be used in hu-man or veterinary medicine to treat (i.e., cure, alleviate or prevent) a variety of conditions. They are useful as antiinflammatory agents, chemopreventive agents to prevent oncogenesis and tumor promotion, protective agents to reduce cytotoxic and cardiotoxic effects of anticancer drugs or protect ischemic tissue. The treatment of .,~

medical conditions with SOD is referred to as "SOD
therapy". Like native SOD, the analogs catalyze the dismutation of superoxide radicals to hydrogen peroxide and molecular oxygen and may thus be used to reduce perfusion injury following ischemia, prolong the viability of excised isolated organ transplants, reduce injury on reperfusion following organ transplant or spinal cord ischemia, reduce cardiac infarct size, reduce spinal cord injury and treat bronchial pulmonary dysplasia.
For therapeutic medical applications the analogs may be administered orally or parenterally to individuals in various dosage forms such as tablets, capsules and injectables. When used to treat tissues in vitro the analog will be added to the perfusion or culture medium.
The analog may be administered neat or admixed in effec-tive amounts with pharmaceutically acceptable solid, semisolid or liquid vehicles such as albumins, globulins, dextran, Ficoll polymers, sugars, starches and liposomes.
Preferably the SOD analog is conveniently stored lyophilized with sugar, usually sucrose, usually in a ratio of 1:2 w/w. The lyophilized enzyme is conveniently reconstituted in a suitable diluent for the particular application. For example, to treat inflammatory joint disease the SOD analog may be reconstituted in physiologic saline in a volume convenient for intraarticular administration.
The dose of SOD analog administered to an individual will depend upon the nature of the individual being treated, the mode of treatment and the condition being treated. In general the amount administered must be sufficient to provide an enzymatically effective amount of the mutein at the desired site of treatment. In this regard, when the analog is administered systemically, larger doses will typically be required than when the analog is administered locally at the site that requires treatment. By way of example, human patients having in-flammatory joint disease are treated by a weekly intraarticular injection into a joint afflicted with the disease of a solution having SOD analog in a suitable diluent in an amount effective to reduce inflammation, usually 1 to 10 mg, more usually 2 to 6 mg. The injec-tions are given weekly for a period of time sufficient to reduce inflammation, usually for 2 to 8 weeks, more usu-ally for 4 to 6 weeks. When used to minimize post-ischemic tissue damage the human patient is administered 10 mg to 1,000 mg, more usually 50 mg to 500 mg of SOD
analog in a suitable diluent during the ischemic reaction.
When the patient suffers ischemia due to a disease the solution is administered intravenously or intraarterially as a bolus dosage or a continuous infusion. In such situations, the SOD analog may be administered in conjunc-tion with fibrinolytic agents such as urokinase, streptokinase or tissue plasminogen activator (tPA). When ischemia damage is due to a surgical procedure, SOD analog is administered during surgery. This application finds particular use in organ transplant surgery where SOD is preferably administered prior to reirrigation of the organ and is also useful in any other surgery where bloodflow to an organ is interrupted, such as open heart surgery.

F. Examples The invention is further exemplified by the fol-lowing examples. These examples are not intended to limit the invention in any manner.

Example 1: Construction of Expression Vectors for Produc-ing SOD-AS-T-2 This example describes the construction of expression vectors for producing a SOD analog that has four amino acid substitutions located in loop II and two amino acid deletions (see Fig. 2). The substitutions were as follows (single letter residue designations followed by residue position number (native Cu/Zn numbering); -indicates a deletion).

Native Sequence K23 E24 S25 N26 G27 P28 Analog Sequence R G D - - T

The analog is designated SOD-AS-T-2 and the bacterial and yeast expression vectors used to produce the analog are designated pSOD-AS-T-2 and pYSOD-AS-T-2 respectively.
The constructions of plasmids pSOD-AS-T-2 and pYSOD-AS-T-2 are depicted in Figure 3. The ampicillin-resistant plasmid p6610SC4 is prepared by inserting the gene segment shown in Figure 1 between the NcoI and SalI
sites of plasmid pNco5AHSODm, a diagram of which is shown in Figure 8 and the construction of which is depicted in Figure 9. A synthetic gene segment (Figure 4) prepared from synthetic oligomers designated T-2(u)(78) and T-2(1)(82) by phosphorylating individual oligomers and an-nealing them (by slow cooling of an equimolar solution from 70 to 25C), was inserted between the ApaI and StuI
2S sites of p6610SC4 to yield plasmid pSOD-AS-T-2. Plasmid pSOD-AS-T-2 was then treated with NcoI and SalI and the resulting digest was ligated with the kanamycin-resistant, ampicillin-sensitive vector, pPGAPXSSB-bovine which had been digested with NcoI and SalI and treated with alkaline phosphatase. Following ligation and transformation, plasmid pPGAPXSSB-SOD-AS-T-2 was selected on kanamycin.
The construction of pPGAPXSSB-bovine used above was as follows: between the NcoI and SalI sites of pPGAPXSSHSODm (see Figures 10 and 11) was inserted a segment encoding bovine Cu/Zn SOD. Kanamycin resistance was then conferred by inserting at the PstI site the approximately 1200 bp fragment carrying the kanamycin-resistance element obtained from a PstI digest of PUC-4K (Pharmacia). A SacI
digest of pPGAPXSSB-SOD-AS-T-2 was then ligated with a phosphatase-treated SacI digest of the ampicillin-resistant vector pCl/lXSS. The resulting plasmid, pYSOD-AS-T-2, was selected on ampicillin.
The constructions of plasmids pNco5AHSODm, pPGAPXSSHSODm, and pCl/lXSS were as follows.
Plasmid pNco5AHSODm was constructed as depicted in Figure 9. An EcoRI fragment of about 850 bp from a lambda clone coding from the pre-human Mn SOD was excised by EcoRI digestion and purified by agarose gel electrophoresis. This fragment was ligated to ptac5 (Hallewell et al., Nucleic Acids Res (1985) 13:2017) previously digested with EcoRI. The EcoRI site is present in the polylinker of ptac5. The ligation mix was used to transform _ coli. Several transformant colonies selected in ampicillin-L broth plates were screened by restriction analysis. One clone (ptac5HSODm) containing the correct orientation (3'-end near the SalI site on the polylinker) was selected for further manipulation.
Plasmid ptac5HSODm was digested with NarI, which cuts in the Mn SOD cDNA sequence coding for amino acids 12-13.
Synthetic linkers of the sequence described in Figure 9 were ligated to linearized ptac5HSODm. The linkers provide for an NcoI overhang, a mini-gene coding for 4 amino acids which incorporates in its sequence a Shine-Dalgarno sequence for ribosome binding, a stop signal, an ATG for the first translation initiation methionine codon for human Mn SOD and a sequence coding for ten amino acids of the human Mn SOD.
The first two amino acids of the mature protein were not included in the synthetic linker. Instead, the residue that is in third position in the mature protein (Ser) is adjacent to the Met in the linker sequence. This choice is made because bacteria and yeast process and cleave after methionine when followed by a serine (Tsunasawa et al., J.B.C. (1985) 260:5382). Therefore, a mature human Mn SOD is obtained without an N-terminal methionine. If the initiating methionine is followed by Lys, the first residue of the mature protein, the methionine would not be cleaved. In this case one would obtain a methionyl-human Mn SOD which could be antigenic for human use because it contained an N-terminal methionine.
The ptac5HSODm with ligated linkers was digested with NcoI and SalI (which cuts after the 3'-end of the insert). The NcoI-SalI fragment (ca. 715 bp) was gel purified. This fragment was cloned into NcoI-SalI-digested pSODNco5A to yield pNco5AHSODm.
Plasmid pNco5ASOD is a pBR322-derived bacterial expression plasmid for human Cu/Zn SOD. The plasmid contains the tac promoter and human Cu/Zn SOD cDNA as an EcoRI-SalI insert substituting pBR322 sequences between EcoRI and SalI. The tac promoter is proximal to the EcoRI
site and the direction of transcription is clockwise. The tac promoter and human Cu/Zn SOD cDNA insert of pSODNco5A
was obtained from pNco5ASOD (Hallewell et al., supra).
Plasmid pPGAPXSS, used to construct pPGAPXSSHSODm, is a derivative of pPGAP (EPO 164 556), in which the following linker was inserted at the junction between the GAP promoter and/or GAP terminator sequences and the vector sequences:
BqlII SacI SacII XhoI BamHI
BXSSBB-U 5' GATCTG AGCTICC GCIGGCITCGA G¦GATC CA 3 BXSSBB-L 3' AclTcGA GG¦CG CCG AGCTIC CTAGIGTCTAG 5' The linkers provide for SacI, SacII, XhoI and BamHI
adjacent to both 5'-end of GAP promoter sequences and 3'-end of GAP terminator sequences. Both BamHI and ~
sites coming from pPGAP end and linkers, respectively, were not reconstructed.
PolyA sequences were removed from pNco5AHSODm by digesting the plasmid with PvuI, which cuts upstream from the polyA tract. The overhang was filled in using the Klenow fragment of DNA polymerase I. The plasmid was subsequently digested with NcoI. A 615 bp fragment containing the hSODm cDNA was gel isolated and was reinserted into pNco5AHSOD, digested with NcoI and SmaI
(which cuts next to SalI, as indicated in Figure 1) to yield pNco5AHSODm-PA, after transformation of E. coli MC1061.
Plasmid pNco5AHSODm-PA is linearized with NarI
and ligated to the synthetic DNA linker shown below. The lower strand of the linker is phosphorylated in this liga-tion.
Met Ser Leu Pro Asp Leu Pro Tyr Asp Tyr Gly 5' ATG TCT TTG CCA GAC TTG CCA TAT GAC TAC GG 3' 3' AGA AAC GGT CTG AAC GGT ATA CTG ATG CCG C 5' Following ligation, the plasmid is cut with SalI and the about 720 bp linker to SalI fragment is isolated by preparative agarose gel electrophoresis. This fragment is ligated to NcoI- and SalI-cut pPGAPXSS in the presence of N I. After transformation of MC1061, a colony containing the recombinant plasmid pPGAPXSSHSODm is obtained.
Plasmid pCl/1 (see EPO 116 201) was linearized with BamHI and phosphatased. The synthetic DNA linkers of sequence shown below, having BamHI complementary ends and restriction endonuclease sites for SacI, SacII and XhoI, were phosphorylated with T4 polynucleotide kinase. After removal of the kinase, they were ligated to the linearized pC1/1. The BamHI is not reconstituted by ligation of this linker.
5' GATCG AGCTtCCC GCIGGCITCGA GC 3' 3'C¦TCGA GGG~CG CCG AGCTICGCTAG 5' SacI SacII XhoI
After transformation of the E. coli strain MC1061, a re-combinant plasmid, pCl/lXSS, was obtained which had the linker inserted at the BamHI site. SacI, SacII and XhoI
are unique sites in pCl/lXSS.

Example 2: Construction of Expression Vectors for Produc-ing SoD-As-T-8 This example describes the preparation of expression vectors for producing a SOD analog that has a C-terminal fibrinogen adhesive domain extension. The sequence of the extension was as follows:

Native Sequence G150 I151 A152 Q153 Analog Sequence G I A Q GQQHHLGGAKQAGDV

In the extension GQQHH is a fibrinogen-derived spacer sequence and the remaining decapeptide is the adhesive domain.
This analog is designated SOD-AS-T-8 and the expression plasmids used to produce it are designated pSOD-AS-T-8 for bacteria and pYSOD-AS-T-8 for yeast.
The construction of plasmid pYSOD-AS-T-8 is depicted in Figure 5. A gene segment (Figure 6) prepared from synthetic oligomers designated T-8(u)(87) and T-8(1)(87) by phosphorylating individual oligomers and an-nealing them (by slow cooling of an equimolar solution from 70 to 25C), was inserted between the BamHI and SalI
sites of p6610SC4 to yield plasmid pSOD-AS-T-8. Plasmid pSOD-AS-T8 was then treated with NcoI and SalI and the resulting digest was ligated with the kanamycin-resistant, ampicillin-sensitive vector, pPGAPXSSB-bovine, which had first been treated with NcoI and the alkaline phosphatase.
Following transportation, plasmid pPGAPSXXB-SOD-AS-T-8 was selected on kanamycin. A SacI digest of pPGAPXSSB-SOD-AS-T-8 was then ligated with phosphatase-treated SACI digest of the ampicillin-resistant vector, pCl/lXSS. The resulting plasmid pYSoD-AS-T-8 was selected on ampicillin.

Example 3: Construction of Expression Vector for Producing This example describes the preparation of a yeast expression vector for producing an analog of a SOD polymer that has the fibrinogen adhesive domain extension described in Example 2. This analog is designated SOD-H-T8 and the expression plasmid used to produce it is designated pYSOD-H-T8.
Plasmid pPGAPSODHAl-met (described in EPA 0 283 244 published 21 September 1988), was digested with ApaI and SalI
and then treated with alkaline phosphatase. The larger vector fragment was then gel-isolated. Also prepared was an ApaI-S I cassette, carrying a portion of the gene coding hSOD-AS-T-8, by digestion of pYSOD-AS-T-8 with ApaI and SalI, followed by gel-isolation of the ca. 500 bp fragment. This fragment was cloned into the vector described above to yield pPGAPSoD-H-T8. Plasmid pPGAPSOD-H-T8 was then partially digested with BamHI and the ca. 2443 bp gel-isolated fragment was cloned into a vector which had been derived from pCl/l by BamHI digestion and treatment with alkaline phosphatase.
Plasmid pYSOD-H-T8 was obtained.

- ~ 335577 Example 4: Expression of SOD-AS-T-2, SOD-AS-T-8 and SOD-E. coli strain MC1061 was transformed with plasmids pSOD-AS-T-2 or pSOD-AS-T-8 and selected on ampicillin. Transformants were then grown overnight at 37C in 1.5 ml of L-broth containing 100 mg/ml of ampicillin and 1.0 mM CuSO4. To the cell pellet obtained by centrifugation was then added 60 ul of lysis buffer (50 mM Tris, pH 8.0, 1.0 mM phenylmethylsulfonyl fluoride, 2.0 mM CuSO4) and 50 ul of glass beads. The mixture was vortexed 10 sec and shaken (30 min, Eppendorf*Mixer 5432).
Twenty ul of the supernatant obtained by centrifugation (7 min) was mixed with 7 ul of 6 x sample buffer (30%
glycerol and dyes), loaded on a 10% polyacrylamide gel and electrophoresed. Gel staining for enzymatic activity was then performed according to Beauchamp and Fridovich (Analyt. Biochem. (1971) 44:276-187). Both are active.
S. cerevesiae, strain PO17, was transformed with pYSOD-AS-T-2, pYSOD-AS-T-8 or pYSOD-H-T8. Transformants were grown and assayed for intracellular recombinant SOD
activity as described in EPA 0138111. Each analog exhibited enzymatic activity.
Ten liters of cell culture of the above yeast transformants were grown. Cells were lysed, and SOD-AS-T-2, SOD-AS-T-8 and SOD-H-T8 were purified as described for recombinant Cu/Zn SOD in said EPA 0131811.

Example 5: In Vitro Platelet Binding Assay The ability of an analog to bind to platelets may be determined as follows. Three-tenths mg to 30 mg of test SOD analog or rhSOD (recombinant human Cu/Zn SOD) is added to 1.0 ml of heparinized or unheparinized blood and to 1.0 ml of plasma fractions prepared from heparinized and unheparinized blood. After 15 min incubation, the whole blood is centrifuged and SOD activity of the super-(*) Trademark ~f.i~ ~

natant is determined (pyrogallol assay). Enzymatic activ-ity is also determined for plasma fractions to which SOD
or its analogs is similarly added. A decrease in the ratio of enzymatic activity in the whole blood supernatant vs treated plasma compared to the ratio derived from rhSOD-treated fractions (i.e., the control) reflects an increase in platelet binding of the test analog over that of rhSOD.
In preliminary tests using this technique, SOD-AS-T-2 exhibited a decrease in said ratio relative to rhSOD, whereas SOD-AS-T-8 exhibited a ratio approximately the same as rhSOD.
In addition to SOD-AS-T-2, SOD-AS-T-8, and SOD-H-T8 applicants have attempted to prepare or have prepared other SOD analogs in bacteria that included adhesive peptide signals but did not exhibit enzymatic activity.
Some of these analogs are reported in the table below.

Modification Location Sequence Loop III
Wild: G37 L38 T39 E40 G41 L42 Analog: RGD (fibronectin insertion) Analog: GRGDS (fibronectin insertion) Greek key Loop Wild: L106 S107 G108 D10~ H110 Clll Analog: R
C-Terminus Wild: G150 I151 A152 Q153 Analog: " " " " GQQHHL-GYAVTGRGDSPASSKPIS
(fibronectin extension using its GRGDS complete loop and a tolerated fibrinogen spacer) C-Terminus Wild: G150 I151 A152 Q153 Analog: " V C G PGLWERQAREHSERKKRRRESECKAA
(C-terminal extension with EC-SOD tail and 3 "internal" Cu/Zn SOD residues altered) Analog: " I A Q GQQHHLC W GVCGPGLWERQAR-EHSERKKRRRESECKAA
(a longer EC-SOD tail including all 3 Cys with a fibrinogen spacer; "internal" amino acids unaltered) Modifications of the above-described modes for carrying out the invention that are obvious to those of skill in the fields of protein chemistry, genetic engineering, pharmaceuticals, medicine and related fields are intended to be within the scope of the following claims.

Claims

1. A superoxide dismutase analog having dismutase activity and whose amino acid sequence includes a heterologous peptide domain that provides the analog with an ability to bind to a moiety that localizes at or is local to a body site at which superoxide dismutase therapy is desired or increased corporal lifetime, wherein i) the superoxide dismutase (SOD) is human Cu/Zn superoxide dismutase;
ii) the heterologous peptide domain comprises a RGDX tetrapeptide, and said domain provides the analog with an ability to bind a to a moiety that localizes at or is local to a body site at which SOC therapy is desired;
iii) amino acids 23 to 28 of the native sequence of Cu/Zn superoxide dismutase is modified as follows to include the RGDX tetrapeptide:
Native sequence: K23E24S25N26G27P28 Mutant sequence: R23G24D25- - T26 where "-" represents an amino acid deletion;
iv) the analog is SOD-AS-T-2 that has increased binding to a platelet.

2. The analog of claim 1 wherein the domain is located in a hydrophilic loop of the superoxide dismutase or at the carboxy terminus of the superoxide dismutase.

3. A pharmaceutical composition for providing superoxide dismutase therapy comprising the analog of claim 1 admixed with a pharmaceutically acceptable parenteral vehicle.

4. A pharmaceutical composition for providing superoxide dismutase therapy comprising the analog of claim 2 admixed with a pharmaceutically acceptable parenteral vehicle.

5. DNA encoding the superoxide dismutase analog of claim 1.

6. DNA encoding the superoxide dismutase analog of claim 2.

7. An expression vector for expressing the DNA of claim 1 in a micro-organism comprising the DNA of claim 1 operably connected to DNA that enables the expression of the DNA in the organism.

8. A host micro-organism containing the vector of claim 7 which permits the expression of the analog by the micro-organism.

9. A method of producing a superoxide dismutase analog having dismutase activity and whose amino acid sequence includes a heterologous peptidedomain that provides the analog with an ability to bind to a moiety that localizes at or is local to a body site at which superoxide dismutase therapy is desired comprising growing the micro-organism of claim 8 in a suitable growth medium under conditions that result in the expression of said DNA encoding the analog and production of said analog.